In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorder osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Our Section has generated a knock-in murine model for OI with a COL1A1 collagen mutation. Recently, we collaborated to investigate the material properties of Brtl cortical and endosteal bone using acoustic transmission microscopy. Collagen orientation in Brtl endosteal bone had a strong reduction in periodically alternating collagen orientation compared to WT. Also in Brtl endosteal bone sound velocity was significantly increased, demonstrating that the predominant effect of the Brtl mutation is on endosteal bone. Using Brtl, we conducted a trial of bisphosphonate, which complements our pediatric trial. Detrimental changes were detected in bone material strength and brittleness. Furthermore, retention of mineralized cartilage disrupts matrix continuity and may contribute to crack propagation. Bone cell function declined and osteoblasts were altered to a flattened morphology, similar to lining cells. These studies contribute cautionary notes regarding elevated cumulative bisphosphonate dose. Brtl is also being used as the model for testing an anabolic therapy for OI, anti-sclerostin antibody (SclAB), which works by stimulating bone formation along the canonical wnt pathway. SclAB was demonstrated to be effective in increasing cortical bone formation in both growing young Brtl mice and in adult mice. Brtl femora increased cortical bone formation and mechanical strength, without exacerbating the underlying brittleness of OI bone material. These data suggest SclAB treatment does not impair material properties. Although SclAB is a short-acting drug, a single dose of bisphosphonate will preserve the gains to trabecular bone mass following cessation of antibody. Concurrent administration of low dose bisphosphonate with anti-sclerostin antibody revealed synergistic effects on trabecular mass and vertebral stiffness. Cortical gains in mass and stiffness occurred through SclAB alone. Thus, minimal antiresorptive treatment may be able to amplify the effects of SclAB, without incurring the detrimental effects of BP on bone material quality. The fibroblasts of OI patients have been utilized to test the effect of the chemical chaperone, 4-PBA, on ER retention of type I collagen and activation of the unfolded protein response. Cells carrying mutations in COL1A1 upregulate autophagy by enhancing the expression of Atg5. Both COL1A1 and COL1A2 mutations are connected to increased cellular apoptosis. Treatment with 4-PBA, increased not only collagen secretion, but also general protein secretion from mutant cells and rescued cell death. Testing of this approach in patient osteoblasts will be important. We identified a novel high bone density form of OI caused by mutations in the C-proteinase cleavage site. The Asp-Ala dipeptide between the telopeptide and the C-propeptide of each chain is cleaved by C-proteinase/BMP1 to release mature collagen. Children with substitutions at these residues present with fractures and a high DEXA z-score. Interestingly, despite the high DEXA, radiographs and histology are similar to type I OI and point to matrix deficiency. Pericellular processing of procollagen C-propeptide is delayed. FTIR and BSEM revealed bone mineral content higher than other forms of OI. These data not only reveal a novel form of OI but also provide new fundamental information on roles of procollagen processing and the mechanism of tissue mineralization. We have now generated a mouse model for HBM OI, to investigate the role of type I procollagen C-propeptide cleavage in the mechanism of increased bone mineralization, both at the matrix and intracellular levels. We have also studied amino acid substitutions in the procollagen C-propeptide itself. These mutations are intriguing because they occur in a region of the promolecule that is not incorporated into matrix, but they cause the full clinical severity spectrum of OI. On immumofluorescence microscopy, procollagen with C-propeptide defects was mis-localized to the ER lumen, in contrast to normal localization at the ER membrane. Pericellular processing of the mutant C-propeptide was defective, as were in vitro cleavage assays with purified BMP1. Our data suggest that the 3D structure of the C-proteinase cleavage site is altered by these mutations. Incorporation of overmodified collagens into matrix is reflected in altered fibril diameter and organization in tissue. In bench studies aimed at understanding the basis of the phenotypic variability of patients with the identical OI-causing mutation, we collaborated on investigations of cellular cytoskeleton in Brtl lethal and surviving mice. Components of intermediate filaments, microtubules and actin filaments were all shown to be abnormal only in tissues from lethal mice. The aberrant cytoskeleton affects TGF-b and integrin signaling. This data was extended to cells from patients with lethal and non-lethal mutations caused by identical glycine substitutions. They point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment. We are continuing our clinical studies of children with types III and IV OI. The BEMB undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both the primary skeletal gains and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. The treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area. The increase in vertebral BMD in the treatment group tapered off after one to two years of treatment. There was no significant change in ambulation level, lower-extremity strength or pain in treated children. Hence the changes previously reported appear to be placebo effects in uncontrolled trials. We recommend that pamidronate treatment of children with types III/IV OI be limited to three years, with subsequent follow-up of bone status. We are currently engaged in a dose comparison trial. We are also focusing on the variability of response to treatment in each group. The improvements in vertebral height and area do not correlate with changes in DXA z-score. These differences may be related to important individual variation in ability to synthesize new bone or to remodel bone. The BEMB has also established the first OI-specific longitudinal growth curves for children with types III and IV OI based on data from 100 children with structural mutations in type I collagen. Short stature is one of the cardinal features of OI. These curves show that the height trajectory of OI children is influenced by both type and gender, but only OI type affected the weight curves. Interestingly, head circumference did not differ by gender or type. Neither length, weight nor head circumference were influenced by the particular mutated chain. This data was also used to derive BMI curves for OI, demonstrating distinct shifts above the CDC curves.